Browsing by Author "Marti, S."
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- Assessment of trimethoprim-sulfamethoxazole susceptibility testing methods for fastidious Haemophilus spp.Publication . Sierra, Y.; González-Díaz, A.; Tubau, F.; Carrera-Salinas, A.; Moleres, J.; Bajanca-Lavado, Maria Paula; Garmendia, J.; Domínguez, M.; Ardanuy, C.; Marti, S.Background: Several discrepancies were found in clinical routine regarding trimethoprim-sulfamethoxazole (SXT) susceptibility determination depending on antimicrobial susceptibility (AST) method used and growth media. We aimed to compare the determinants of SXT resistance with established susceptibility values for fastidious Haemophilus spp., in order to provide recommendations for optimal SXT measurement. Materials/methods: We collected 50 strains each of Haemophilus influenzae and Haemophilus parainfluenzae at Bellvitge University Hospital. SXT susceptibility was tested by microdilution, E-test, and disc diffusion using both Mueller-Hinton Fastidious (MH-F) and Haemophilus Test Medium (HTM) following EUCAST and CLSI criteria respectively. Mutations in folA, folP and additional determinants of resistance were identified in whole-genome sequenced isolates. Results: Strains presented generally higher rates of SXT resistance when grown on HTM than on MH-F, independent of the methodology used (average MIC 2.6-fold higher in H. influenzae and 1.2-fold higher in H. parainfluenzae). The main resistance-related mechanisms were as follows: I95L and F154S/V in FolA; 3 and 15 base pair insertions and substitutions in folP; acquisition of sul genes; and FolA overproduction potentially linked to mutations in -35 and -10 promoter motifs. Of note, 2 of 19 H. influenzae strains (10.5%) and 9 of 33 H. parainfluenzae strains (27.3%) with mutations and assigned as resistant by microdilution were inaccurately considered susceptible by disc diffusion. This misinterpretation was resolved by raising the clinical resistance breakpoint of the EUCAST guidelines to ≤30 mm. Conclusions: Given the routine use of disc diffusion, a significant number of strains could potentially be miscategorised as susceptible to SXT despite having resistance-related mechanisms. A simple modification to the current clinical resistance breakpoint given by the EUCAST guideline for MH-F ensures correct interpretation and correlation with the gold-standard method of microdilution.
- Insights into the population structure and pan-genome of Haemophilus influenzaePublication . Pinto, M.; González-Díaz, A.; Machado, M.P.; Duarte, S.; Vieira, L.; Carriço, J.A.; Marti, S.; Bajanca-Lavado, M.P.; Gomes, J.P.The human-restricted bacterium Haemophilus influenzae is responsible for respiratory infections in both children and adults. While colonization begins in the upper airways, it can spread throughout the respiratory tract potentially leading to invasive infections. Although the spread of H. influenzae serotype b (Hib) has been prevented by vaccination, the emergence of infections by other serotypes as well as by non-typeable isolates (NTHi) have been observed, prompting the need for novel prevention strategies. Here, we aimed to study the population structure of H. influenzae and to get some insights into its pan-genome. We studied 305H. influenzae strains, enrolling 217 publicly available genomes, as well as 88 newly sequenced H. influenzae invasive strains isolated in Portugal, spanning a 24-year period. NTHi isolates presented a core-SNP-based genetic diversity about 10-fold higher than the one observed for Hib. The analysis of key factors involved in pathogenesis, such as lipooligosaccharides, hemagglutinating pili and High Molecular Weight-adhesins, suggests that NTHi shape its virulence repertoire, either by acquisition and loss of genes or by SNP-based diversification, likely towards host immune evasion and persistence. Discreet NTHi subpopulations structures are proposed based on core-genome supported with 17 candidate genetic markers identified in the accessory genome. Additionally, this study provides two bioinformatics tools for in silico rapid identification of H. influenzae serotypes and NTHi clades previously proposed, obviating laboratory-based demanding procedures. The present study constitutes an important genomic framework that could lay way for future studies on the genetic determinants underlying invasiveness and disease and population structure of H. influenzae.